Neutrophils enhance early Trypanosoma brucei infection onset

Abstract

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

Figure 1

Innate immune cell recruitment to the dermal trypanosome infection site. Numbers of (A) neutrophils (CD45⁺CD11b⁺Ly6C^IntLy6G⁺) and (B) monocytes (CD45⁺CD11b⁺Ly6C^HiLy6G⁻) recovered from ear pinnae of C57Bl/6 mice exposed to the bites of naive or T.b.b. SG+ tsetse flies. The gating strategy is shown in Supplemental Fig. S1. Bar charts for each time point are from n = 6/group of infected mice and n = 3 for the uninfected control mice. The kinetics at the various time points are representative of two independent experiments, the neutrophil recruitment at 4.5 hpi and 90 hpi has been confirmed in respectively 7 and 4 independent experiments. Data in the bar charts are the means ± SEM. Statistical significances indicated above the bars are based on the two-way ANOVA with a Tukey multiple comparison test.

Figure 2

Inflammatory gene transcription at the dermal trypanosome infection site. Heat map representing the expression of differentially regulated inflammatory genes in the dermis of mice exposed to SG⁻ tsetse fly bites or bites of T.b.b. AnTAR1 SG⁺ flies at 4.5, 18 and 90 hours post bite exposure. Indicated are the mean log2-fold changes in expression. Individual transcription levels were normalized using the TUBA1A and EEF2 reference genes using the Q base Plus software. Data in the graphs are the means and SEM of 6–12 controls, 6–9 ear tissue samples exposed to naive bites and 6–9 samples exposed to T.b.b. AnTAR1.

Figure 3

In vivo trypanosome phagocytosis in the murine dermis. (A) Flow cytometry analysis of T.b.b. AnTat1.1E^dsRed phagocytosis by white blood cells (CD45⁺/dsRed⁺ cells in the P2 gate) in the dermis of mice infected through the bites of an SG+ tsetse fly. The P1 gate (CD45^dim/dsRed⁺) represents events with a low FSC/SSC indicating parasitic debris rather than bona fide cells. (B) Confocal microphotograph of intradermal uptake of T.b.b. AnTat1.1E^dsRed by LysM-GFP⁺ cells in the infected ear dermis. Uptake occurs if trypanosomes display a reduced motility (see Supplemental Videos S1 and S2). White arrows indicate free living trypanosomes, whereas blue arrows show phagocytosed trypanosomes in the dissected ear tissue.

Figure 4

Effect of neutrophil depletion on parasitemia progression in a susceptible mouse model. Parasitemia progression in TNF-α deficient C57Bl/6 mice following a single intraperitoneal injection at day −1 of the depleting antibodies 1A8 (A) or RB6-8C5 (B) and the appropriate antibody isotype controls (2A3 or LTF-2). Parasitemia data are the means ± SEM of n = 6 mice/group. Data are representative of two independent experiments. Statistical significance levels based on the Mann-Whitney test are indicated.

Figure 5

Effect of congenital neutropenia on trypanosome infection progression in Genista mice. Flow cytometry analysis on neutrophil and monocyte counts in peripheral blood of (A) heterozygous (controls) and (B) homozygous Genista mice. (C) Parasitemia progression in heterozygous (n = 6) and homozygous (neutropenic, n = 9) Genista mice following a tsetse mediated infection with T.b.b. AnTAR1. Parasitemia data are the means ± SEM. Data are representative of 2 independent experiments. Statistical significance levels based on the Mann-Whitney test are indicated.

Figure 6

Effect of congenital neutropenia on the dermal trypanosome infection progression in Genista mice. BLI analysis of dermal parasite burdens following a T. brucei AnTat1.1E^PpyRE9 infective tsetse fly bite. (A) Comparison of the in vivo biodistribution and dermal burdens in heterozygous (+/−, n = 3) and homozygous (−/−, n = 3) Genista mice subjected to a dorsal and ventral image analysis. (B) Luminescence quantified within a ROI corresponding to the left ear, where parasites were inoculated by a tsetse fly bite. Luminescence was measured using a 3 minute exposure time between 3 and 6 dpi. From 7 dpi onwards, 5 second exposure times were used to avoid saturation. No statistically significant differences were recorded. NA: one animal succumbed within 12 days of infection, precluding image acquisition.